The impact of saliva collection and processing methods on CRP, IgE, and Myoglobin immunoassays

Abstract

Background Owing to its ease of collection, saliva is potentially the sample of choice in diagnosis. Salivary biomolecules have provided a porthole in surveying a person's health and well‐being. Our study aims were (1) to demonstrate the effects of pre‐analytical steps, collection and pre‐processing techniques on salivary protein detection and (2) to establish an indication of salivary reference intervals for 3 biomolecules of clinical interest. Methods Saliva samples were collected from participants (n = 25, ages 20–35 years) using the following methods: no stimulation (resting/unstimulated), mechanical, and acid stimulation. The saliva was prepared for analysis by: unprocessed, post standard centrifugation in a container without any additives, and centrifugation using Centrifugal Filter Unit (Amicon® Ultra‐0.5). AlphaLisa® assays were used to measure the levels of C‐Reactive Protein (CRP), Immunoglobin (IgE) and myoglobin in saliva samples. Results Saliva flow rates were lowest with the resting/drooling collection method. The lowest total protein concentration was with acid stimulation. Unstimulated and mechanically stimulated collections produced no effect on the CRP and IgE levels while myoglobin levels were highest with the unstimulated collection. Acid stimulation had a negative impact on the measured concentrations of IgE and myoglobin (except for CRP levels). Conclusion Mechanical stimulation was the most viable option for collecting saliva without affecting the levels of CRP and myoglobin. The processing methods had an adverse effect on the concentration of total protein as well as on CRP and IgE concentrations.

Circulating Fragments of N-Terminal Pro–B-Type Natriuretic Peptides in Plasma of Heart Failure Patients

“…Therefore, monoclonal antibodies that target the nonglycosylated sites between NTproBNP 28 -45 [i.e., NT-proBNP 30 -35 (Glu-Leu-GlnVal-Glu-Gln motif)] are likely to exclude the effects of O-glycosylation on the measurement of plasma NTproBNP. This, together with monoclonal antibodies targeting NT-proBNP [13][14][15][16][17][18][19][20] , is likely to provide a more standardized measurement of the plasma NT-proBNP for future "sandwich" immunoassays. Our data indicated that although immunoreactive NT-proBNP was present in human plasma as multiple fragments of the full-length NT-proBNP molecule, specific epitopes of the peptide appear to be more abundant, providing ideal targets for developing the next generation diagnostic assays for clinical use.…”

“…The NTproBNP standards were prepared using pooled plasma collected from healthy volunteers (n ϭ 10) and mixing it with the High Block immunoassay buffer in a ratio of 50%:50%. This enabled us to overcome the matrix (A), The 6 immunoassays use diagnostic-grade monoclonal antibodies to detect NT-proBNP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] , NT-proBNP , NT-proBNP , NT-proBNP 28 -76 , NT-proBNP , and NT-proBNP . N-and C-terminal proteolytic sites detected by our MS analysis are shown with red vertical lines.…”

“…The 6 NT-proBNP immunoassays are named according to the first amino acid that the capture antibody binds to on the NT-proBNP molecule and the last amino acid that the detection antibody binds to on the NT-proBNP molecule of 1-76 amino acids: NT-proBNP 1-20 (5B6 1-12 and 13G12 [13][14][15][16][17][18][19][20] ); NT-proBNP (18H5 [13][14][15][16][17][18][19][20] and 11D1 28 -45 ); NTproBNP (5B6 [1][2][3][4][5][6][7][8][9][10][11][12] and 11D1 28 -45 ); NT-proBNP 28 -76 (11D1 28 -45 and 28F8 67-76 ); NT-proBNP (18H5 [13][14][15][16][17][18][19][20] and 28F8 67-76 ); NTproBNP 1-76 (5B6 [1][2][3][4][5][6][7][8][9][10][11][12] and 28F8 67-76 ). Each NTpr...…”

High-Yield RNA-Extraction Method for Saliva

“…All participants gave informed consent before sample collection. Whole-mouth saliva was collected from healthy, resting control individuals (n ϭ 9) and patients (n ϭ 8), as previously described (11)(12)(13)(14). Saliva was collected in DNase-and RNase-free Falcon tubes (50 mL), frozen immediately on dry ice, and stored at Ϫ80°C until further analysis.…”

Towards autonomous lab-on-a-chip devices for cell phone biosensing

“…The most common forms of saliva collection include (1) the passive drool technique (Christodoulides et al, 2005), where saliva is accumulated in the floor of the mouth for a defined period of time and then spat into a test tube where some few mL are collected and (2) the absorbent device technique, which uses elements such as swabs to sample the saliva, which can be directly integrated into lateral flow immunoassays, such as the commercial devices used for HIV testing (Dewsnap and Mcowan, 2006;Zelin et al, 2008;Mohamed et al, 2012).…”